The invention relates to veterinary vaccines for prevention of hematophagous infestation of cattle and medical treatment of thrombosis.
Losses in livestock production in the United States due to ectoparasite infestations have been estimated to exceed $2.26 billion annually (Byford et al. (1992) J. Anim. Sci. 70:597-602). Of the five to six major arthropod pest species involved, the horn fly Haematobia irritans linnaeus is the most significant and widespread. Its annual economic impact on cattle production in the U.S.A. has been estimated at $730.3 million. In Canada, control of this ectoparasite in cattle production has been estimated to reduce losses by $71-107 million per year using 1977 dollar values (Haufe and Weintraub (1985) Can. Entomol. 117:901-907). Thus in North America, the annual economic impact on cattle production by this blood-sucking fly approaches $1 billion.
Physiological manifestations of hornfly infestation include an increase in heart rates, respiration rates, and rectal temperatures. Additionally, water consumption and urine production are significantly increased as well as urinary nitrogen secretion. Blood cortisol concentrations are also significantly increased. Decreased weight gain, increased activity, and decreased grazing have also been reported. (Schwinghammer et al. (1986) J. Econ. Entomol. 79:1010-1014).
The adult stage of both sexes of H. irritans are obligate ectoparasites that blood-feed intermittently during the 24 hours of the day. Unlike other dipterous pests that are transient blood-feeders, (black flies, mosquitoes, horse flies, stable flies), the winged adults of H. irritans remain on the bovine host and, when needing nourishment, recurrently insert their mouthparts into the skin to feed. Harris et al. (1974) Ann. Entomol. Soc. Am. 67:891-894, noted that under experimental conditions, female horn flies spent an average of 163 minutes/day feeding; males averaged 96 minutes per day. Each female ingested an average of 17.1 mg of blood per day while males imbibed 12.1 mg/individual due to the difference in feeding times (Harris and Frazer (1970) Ann. Entomol. Soc. Am. 63:1475-1476).
The scientific literature describing the salivary gland physiology of H. irritans, particularly with reference to blood-feeding, is sparse. Hori et al. (1981) Appl. Ent. Zool. 16:16-23, has compared several categories of digestive enzymes in the gut and salivary glands of H. irritans with Stomoxys calcitrans (Linnaeus), the stable fly. Weak aminopeptidase activity was detected in H. irritans saliva, suggesting that proteases and glycosidases in the gut are exclusively responsible for digestion of blood.
The horn fly Haematobia irritans linnaeus is a subspecies with H. i. exigua de Meijere, the buffalo fly that occurs in Australia and elsewhere in the southern hemisphere. Kerlin and Hughes (1992) Med. Vet. Entomol. 6:121-126, have compared enzymes in the saliva of four parasitic arthropodsxe2x80x94H. irritans exigua, Boophilus microplus (Canestrini), Aedes aegypti (Linnaeus), and Lucilia cuprina (Wiedemann) and noted differences in enzyme profiles of saliva between the four species that apparently reflect their dissimilar feeding strategies. These differences were mainly in the type and levels of glycosidase and protease activities. H. irritans exigua saliva, collected by serotonin stimulation and then evaluated by SDS polyacrylamide gel electrophoresis, produced 7-8 bands by silver staining. Apyrase activity in saliva and salivary gland extracts (SGEs) of this species was marginally detectable, suggesting that this subspecies does not prevent bovine platelet aggregation in the same way as many other blood-feeding arthropods (Ribeiro (1987) Ann. Rev. Entomol. 32:463-478).
Furthermore, investigation of immune response of cattle exposed to H. irritans exigua showed production of high levels of circulating antibodies to some but not all of the buffalo fly antigens; nevertheless, flies feeding on previously exposed cattle did not exhibit higher mortality than those fed on unexposed cattle. (Kerlin and Allingham (1992) Vet. Parasitol. 43:115-129).
Elucidation of biochemical strategies adopted by blood-feeding arthropods has advanced in the past decade. Although the presence of anticoagulants in saliva of hematophagous arthropods has been known for at least eight decades, only recently have some of the active components been purified and their molecular structures defined. It has become apparent that coagulation factors such as factors Xa and thrombin (factor II), which occur at a nexus in the coagulation cascade, are frequently targeted.
Studies of saliva from several species of black flies have suggested that specific enzyme targets may be associated with host selection (Abebe et al. (.1994)). For example, data for zoophagic species that prefer cattle indicate that thrombin is an important target molecule whose inactivation may also prevent irreversible platelet aggregation in addition to impeding the coagulation cascade. See Hudson (1964) Can. J. Zool. 42:113-120, for Stomoxys calcitrans; and Parker and Mant (1979) Thrombos. Haemostas (Stuttg.) 42:743-751, on G. morsitans (Westwood) saliva.
Because of the adverse impact of the above-described ectoparasitic infestation in cattle, there is a therapeutic and economic need for preventing such infestation.
There is also need for treatment of thromboembolic diseases. Thromboembolic diseases are among the most important circulatory diseases. A thrombus is a blood clot that partially or completely blocks blood flow through a blood vessel. An embolus is a thrombus that has formed elsewhere in the body, broken free, and traveled to the site where blockage occurs. Blockage in the brain results in a stroke, i.e., a cerebral infarction, a localized area of dead cells. An embolus in a lung can produce pulmonary embolism, one of the principal lung diseases in bed-ridden patients. Bed ridden and elderly persons are also particularly prone to thrombophlebitis, which is a blockage of circulation in a leg caused by an embolus. An embolus or thrombus lodging in one of the blood vessels serving the heart causes necrosis of part of the heart tissue, a myocardial infarction, commonly called a heart attack.
The initiating event of many myocardial infarctions is the hemorrhage into atherosclerotic plaques. Such hemorrhage often results in the formation of a thrombus (or blood clot) in the coronary artery which supplies the infarct zone. This thrombus is composed of a combination of fibrin and blood platelets. The formation of a fibrin-platelet clot has serious clinical ramifications. The degree and duration of the occlusion caused by the fibrin-platelet clot determines the mass of the infarct zone and the extent of damage.
The formation of fibrin-platelet clots in other parts of the circulatory system may be partially prevented through the use of anticoagulants, such as heparin. Unfortunately, heparin has not been found to be universally effective in preventing reocclusion in myocardial infarction victims in which the degree of blood vessel occlusion is greater than or equal to 70%, particularly in those patients with severe residual coronary stenosis. Among the more promising of the agents are hirudin and its analogs, which bind to and inactivate thrombin. Hirudin has a theoretical advantage over heparin as an anti-thrombotic agent. Thrombin bound to thrombi or platelets is relatively protected from inhibition by heparin while hirudin, at least in vitro, is still effective. Other promising investigational agents include fibrinogen receptor antagonists, which block platelet aggregation and dense granule release by a mechanism distinct from that of aspirin, and inhibitors of thromboxane production.
There is therefore a need for additional antithrombin agents which exhibit low toxicity, little or no antigenicity, and a very short clearance time from circulation.
Isolated proteins with antithrombin activity and nucleotide sequences encoding the proteins are provided. The protein named thrombostasin is isolated from the salivary glands of Haematobia irritans, the blood-feeding horn fly. The provided proteins and nucleotides are particularly useful as veterinary vaccines in prevention of blood-feeding in cattle by the infesting horn fly.
The proteins of the invention are also useful in treatment of thrombosis.
Methods of administering the proteins and nucleotide sequences of the invention are also provided.